Tissue printing: splenic red pulp macrophages of once-malaria infected mice are transcriptionally identical to prenatally seeded red pulp macrophages from uninfected mice.

Acute malaria infection with P. chabaudi obliterates embryonically seeded tissue-resident red pulp macrophages in the spleen of C57Bl/6J mice - regardless of whether the infection is mild (mosquito transmitted P. chabaudi AS - no hyperparasitaemia, no measurable clinical manifestations of disease other than low-grade anaemia) or severe (mosquito transmitted P. chabaudi AJ - acute hyperparasitaemia, severe anaemia, hypothermia and prostration). Red pulp macrophages return 100 days later, once mice cleared parasitaemia. We then flow sorted 10,000 red pulp macrophages (lineage-, autofluorescent, F4/80+, B220-, CD11bint, CD11cint) directly into Trizol, extracted total RNA and analysed their transciptome using the affymetrix mouse exon 1.0 ST array. Red pulp macrophages from mice once infected with mild AS or severe AJ P. chabaudi parasites were compared to uninfected age-matched mice. We uncover that red pulp macrophages isolated from the spleens of once-malaria infected mice are transcriptionally identical to prenatally seeded red pulp macrophages from uninfected mice. The spleen tissue niche thus imprints an identical functional profile onto these cells - regardless of their origin. C57Bl/6J mice were bred and housed in individually ventilated cages under specific pathogen free conditions (22°C, 50% humidity) at the University of Edinburgh, UK. Malaria infections were initiated when mice were approximately 9 weeks of age by intravenous injection of 200 sporozoites (according to our previously published protocol: Spence, P.J., et al., Mosquito transmission of the rodent malaria parasite Plasmodium chabaudi. Malar J, 2012. 11: p. 407). We mosquito transmitted two distinct Plasmodium chabaudi chabaudi clones, which we obtained from the European Malaria Reagent Repository (malariaresearch.eu) at the University of Edinburgh: P. chabaudi AS (28AS11) and P. chabaudi AJ (96AJ15). Anopheles stephensi mosquitoes (strain SD500) were reared in house at the University of Edinburgh. We initiated all experimental infections with sporozoites, since we have previously shown that mosquito transmission resets expression of the large sub-telomeric multi-gene families that control parasite virulence, and in turn shapes the host immune response during the pathogenic blood-stage of malaria infection (Spence, P.J., et al., Vector transmission regulates immune control of Plasmodium virulence. Nature, 2013. 498(7453): p. 228-31). 100 days after infection, once mice had cleared parasitaemia, red pulp macrophages were flow sorted from the spleens of mice once-infected with P. chabaudi AS (mild acute infection, n = 4) or P. chabaudi AJ (severe acute infection, n = 3) and their transcriptome compared to red pulp macrophages isolated from age-matched uninfected mice (n = 5).

约氏疟原虫（P. chabaudi）急性疟疾感染可清除C57Bl/6J小鼠脾脏中胚胎定植的组织驻留红髓巨噬细胞（tissue-resident red pulp macrophages），无论感染为轻度（经蚊子传播的约氏疟原虫AS株——无高虫血症，仅伴轻度贫血，无其他可检测到的临床疾病表现）还是重度（经蚊子传播的约氏疟原虫AJ株——急性高虫血症、重度贫血、体温过低与全身衰竭）。待小鼠清除虫血症后，红髓巨噬细胞会在100天后恢复至正常水平。我们随后将10000个红髓巨噬细胞（谱系阴性、自发荧光阳性、F4/80+、B220-、CD11bint、CD11cint）直接分选至Trizol试剂中，提取总RNA，并通过Affymetrix小鼠外显子1.0 ST芯片分析其转录组。将曾感染轻度AS株或重度AJ株约氏疟原虫的小鼠的红髓巨噬细胞，与同龄未感染对照小鼠进行比对。

我们揭示，曾罹患疟疾的小鼠脾脏中分离得到的红髓巨噬细胞，其转录谱与未感染小鼠的胚胎定植红髓巨噬细胞完全一致。由此可见，脾脏组织微龛（spleen tissue niche）会为这些细胞赋予完全相同的功能特征，无论其来源如何。

C57Bl/6J小鼠于英国爱丁堡大学的特定无特定病原体（specific pathogen free, SPF）级独立通风笼具中繁育饲养，环境温度为22℃，相对湿度为50%。小鼠在约9周龄时通过静脉注射200个子孢子（sporozoite）启动疟疾感染，实验遵循我们此前发表的方案：Spence PJ等. 啮齿类疟原虫约氏疟原虫的蚊子传播[J]. 疟疾杂志（Malar J）, 2012, 11: 407.

我们从爱丁堡大学的欧洲疟疾试剂库（European Malaria Reagent Repository，malariaresearch.eu）获取了两种不同的约氏疟原虫克隆：约氏疟原虫AS（28AS11）和约氏疟原虫AJ（96AJ15），并通过蚊子进行传播。斯氏按蚊（Anopheles stephensi，SD500株）在爱丁堡大学内自主繁育。我们均以子孢子启动所有实验性感染，因为此前我们已证实，蚊子传播可重置控制寄生虫毒力的大型亚端粒多基因家族（sub-telomeric multi-gene families）的表达，进而在疟疾感染的致病性血液阶段塑造宿主免疫应答（Spence PJ等. 媒介传播调控疟原虫毒力的免疫控制[J]. 自然, 2013, 498(7453): 228-231）。

感染后100天，待小鼠清除虫血症后，我们从曾感染约氏疟原虫AS株（轻度急性感染，n=4）或AJ株（重度急性感染，n=3）的小鼠脾脏中流式分选红髓巨噬细胞，并将其转录组与同龄未感染小鼠（n=5）分离得到的红髓巨噬细胞进行比对。